Structural Studies on the Translocation of Colicin E3 into Sensitive Cells

Case Western Reserve University, Cleveland OH

Investigators

Abstract

Colicin E3 is a toxic protein secreted by certain strains of E. coli in order to eliminate related strains of bacteria living in the same ecological niche. Colicin E3 carries out three functions: it binds to the BtuB receptor on the target cell envelope, it is internalized into sensitive cells across their cytoplasmic membrane, and once inside the cell, it inactivates the protein biosynthetic machinery of the infected cell by nicking 16S ribosomal RNA between A1493 and G1494 at the ribosomal A site. The producing organism is immune to the toxicity of colicin E3 by virtue of an "immunity protein" (IP), which tightly binds to colicin E3 in a 1:1 complex, and thus renders it inactive. The goal of this project is to understand the interaction of colicin E3 with proteins that facilitate its translocation across the outer and inner membranes of E. coli. The research will focus on the crystal structure determination of three ternary complexes: (i) colicin E3, IP and a C-terminal fragment of TolA; (ii) colicin E3, IP and TolB; (iii) colicin E3, IP and the BtuB cell surface receptor. Another part of this work deals with the dynamic aspects of the colicin E3 structure by itself and in the presence of either IP or the Tol proteins or the BtuB receptor. The degree of flexibility, either segmental or global, will be measured by Fluorescence Energy Transfer (FRET). The crystal structure of the colicin E3-IP binary complex suggests the formation of flexible hinges at the domain junctions in the absence of IP. This will be tested by FRET experiments. These studies will shed light on the mechanism of action of this multifunctional protein.